The epitope targeted by PB692 (black bar) is unique to KCNA10 amongst the KCNA protein family members as shown in the ClustalW alignment. Consensus was determined by the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). (B) PB692 was validated through a co-localization assay, in which COS7 cells were transfected with KCNA10-GFP (green) and stained with PB692 (red). Nontransfected cells (arrows) did not express KCNA10-GFP, nor exhibit any signal from PB692. Scale bar represents 20 μm. (C) Immunostaining of wild type P20 organ of Corti shows PB692 signal (green) in multiple cell types, including inner hair cells (IHC) and outer hair cells (OHC), border cells (BC), internal sulcus cells (ISC), and pillar cells (PC) (left panel). However, this signal persisted in Kcna10TM45/TM45 tissues (right panel), indicating that PB692 is non-specific. Rhodamine phalloidin (red) was used to stain actin. Scale bar represents 20 μm.